Communication between adjacent cells through gap junctions (GJs) occurs in nearly every tissue and is fundamental to coordinated cell behavior during development, differentiation, and tissue maintenance. In addition, a growing number of publications indicate that connexins and GJs also play important, yet underestimated functions in physical cell-cell coupling, and in cellular guiding during migration. Cx43-based gap junctional intercellular communication (GJIC) is of particular interest because it is regulated by both physiological and pathological stimuli. While the modulation of GJIC is well documented, surprisingly little is known about how up- or down-regulation of GJIC is actually achieved. GJ channels are known to open and close (gate) in response to physiological parameters;however GJ-based intercellular coupling could also be regulated through altering the number of GJ channels in the plasma membrane (PM). Ultra-structural analyses identified cytoplasmically located, spherical GJ vesicles in situ, leading to the hypothesis that cytoplasmic GJ vesicles (termed annular gap junctions, AGJs) represent internalized GJs. To test this hypothesis, we investigated the spatiotemporal process of GJ internalization in primary vasculature endothelial cells (PAECs), and in cells that transiently or stably expressed fluorescent protein-tagged Cx43. Our studies demonstrate that rapid and efficient internalization of GJ plaques occurs naturally, for example in response to inflammatory mediators such as thrombin and endothelin. Further studies revealed that entire, or large portions of GJ plaques internalize in a ZO-1-mediated, clathrin-dependent process that forms intracellular double-membrane GJ vesicles. These vesicles are then fragmented into smaller vesicles that can be degraded by lysosomal pathways. Based on these results, we hypothesize that GJ internalization may be used widely to efficiently modulate GJIC and cell-cell coupling. To test this hypothesis, we will address three specific questions: (1) Is GJ internalization commonly used to down-regulate intercellular communication and physical cell-cell coupling? (2) How do clathrin and clathrin-associated proteins internalize double-membrane spanning GJs? And (3) which cellular pathway/s degrade/s internalized GJs, and can internalized GJs re-insert into the PM to restore/up-regulate GJIC and physical cell-cell coupling? We will use a comprehensive, integrated experimental approach that includes the expression of fluorescence-tagged Cx43, molecular biology, biochemistry, immunological and functional assays, and temporal high-resolution light and electron microscopic analyses. The physiological role of GJ internalization will be investigated in two endothelial cell systems in which cellular uncoupling/re-coupling occurs naturally: (a) inflammatory mediator (thrombin, endothelin), and (b) VEGF-mediated stimulation. These studies will contribute to our understanding which mechanisms cells use to maintain and modulate intercellular signaling and cell-cell contacts, and will add novel insights into the many evolving functions of endocytic and autophagosomal protein degradation pathways. PUBLIC HEALTH RELEVANCE: Connexin (Cx) family members (of which there are at least 20 in humans) are the protein constituents of gap junction (GJ) channels that provide direct cell-to-cell communication. Cxs are expressed in many different tissues, with Cx43 predominantly found in most tissues. Human mutations in several Cxs (including Cx26, Cx30, Cx30.3, Cx31, Cx32, Cx43, Cx46, and Cx50) are implicated in a number of diseases including neuropathies, deafness, cataracts, skin disorders, and cranio-facial developmental defects, demonstrating the fundamental need of gap junctional intercellular communication (GJIC) at all stages of life, including development, differentiation, and tissue maintenance. Cx43-based GJIC is of particular interest because it is regulated by both physiological and patho-physiological stimuli. While the modulation of GJIC is well documented, surprisingly little is known about how up- or down-regulation of cell-cell communication is actually achieved. Indeed, cells not only need to be able to precisely up or down-regulate the amount of GJIC, but also to internalize all GJs (physically uncouple from their neighbors) if they need to become migratory (development, tissue differentiation, wound healing, etc.) or need to uncouple for other reasons (mitosis, apoptosis, etc.). An understanding of these processes will contribute to our knowledge of how cells maintain and modulate intercellular signaling and cell-cell contacts, and may in future allow us to prevent unwanted GJ internalization/degradation and human diseases that are linked to the mis-regulation of GJIC.